Accurate laboratory analysis of specimens suspected of containing microorganisms is of utmost importance in the fields of medicine and food technology and safety, among others. While techniques have been developed for improving the rapidity and sensitivity of microbiological identification, drugs have been developed for fighting infection in patients, and sanitary conditions for food processing have become mandated by law, it is evident that problems remain.
For example, septicemia, which is the presence of pathogenic microorganisms in the blood, is one of the most serious types of infections encountered. There is unanimous agreement in the medical profession that septicemia is second only to meningitis in terms of serious infections. Even though modern medicine has provided an armament of antibiotics and antifungal drugs, the mortality rate from septicemia is approximately twenty-five percent. Also, when shock accompanies septicemia, the mortality rate increases to over sixty percent. Debilitating diseases, major surgery, administration of immunosuppressive drugs or anticancer medications cause patients to be particularly prone to septicemia. Early diagnosis of the causative agent in conjunction with the use of the appropriate antibiotic therapy is essential in fighting septicemia. Consequently, it is imperative that the physician know as rapidly as possible, not only that the patient has septicemia, but also the identity and/or antibiotic susceptibility of the microorganisms involved. Thus, proper and timely diagnosis of septicemia depends upon very rapid and efficient analysis of the microorganisms in patient's blood. Further, it is necessary during the analysis of the microorganisms in the patient's blood that the blood sample not be contaminated with microorganisms from the hospital environment
Another example of a disorder caused by microorganisms is the presence of pathogenic microorganisms in the urine, which occurs most commonly in infants, pregnant women, patients with obstructive lesions, following the use of instrumentation in the urinary tract (such as catheters), or with urologic diseases affecting micturition. This disorder can result in a localized infection within the bladder or kidneys When confined to the bladder, the infection is usually well controlled by antimicrobial therapy Once the kidneys are infected, however, lesions may continue to progress despite treatment leading to chronic pyelonephritis and septicemia
In the field of food technology, contamination occasionally becomes a problem that endangers human health Contamination of milk, for example, has been known to occur even where a processing step to kill harmful microorganisms is employed because equipment malfunctions, human error, and sometimes mysterious circumstances contribute to processing ineffectiveness. In such cases, rapid and accurate analysis of specimens from the food processing apparatus and the food itself are important in establishing the cause of the contamination so that the process may be remedied. Various techniques are utilized for analysis of microorganisms. Simple quantitative analysis involves determining the number of microorganisms in a given specimen regardless of microorganism identity. Quantitation may be accomplished by introducing a known volume of specimen (perhaps diluted by a known amount in a nutrient broth) onto a nutrient agar and allowing formation of colonies It may be desirable to determine the identity and/or antibiotic susceptibility of the microorganisms found. Analysis to establish microorganism identity and/or susceptibility is usually accomplished by subjecting individual colonies to differentiating media.
In some instances, accurate quantitation as well as identification of particular microorganisms, rather than mere determination whether that particular microorganism is or is not present is highly important. Thus a determination that a specimen is "positive" for microorganisms or "negative" for microorganisms may be insufficient. Rather if the specimen is positive, it may be necessary to know how many microorganisms of a particular species are present in the specimen. It is normal for certain microorganisms to be present in the human mouth and throat at all times, for example. These normal microorganisms, referred to as normal flora, do not generally cause disease in the numbers normally present. However, it is possible for an organism that may be part of the normal flora to proliferate to such an extent that it becomes a disease-causing organism (pathogen). It can be discerned, therefore, that the difference between the normal state of a human throat, for example, and a diseased human throat may be not in the identity of a particular organism that may survive to the time of analysis, but in the numbers of that organism present in the patient's throat. Generally, the bloodstream is sterile. However, transient bacteremia may occur where a few organisms enter the bloodstream through a cut or sore, for example, which is not usually a cause for alarm. Quantitation of microbes in a blood specimen is highly important to distinguish transient bacteremia from septicemia and, perhaps, specimen contamination. While quantitation is of utmost importance in analyzing blood specimens, determining the identity of the microbial pathogen present is also important. Although it may not be necessary to identify a microorganism taxonomically to treat a patient, it may be important to determine microorganism susceptibility to antibiotics so that proper drug therapy may be chosen. This may be done by identifying the organism by genus and species since drug manufacturers often have pre-determined the effectiveness of a drug on particular taxomonic groups. Alternately, testing for drug effect (antibiotic susceptibility) may be accomplished.
In some fluids, microorganism concentration may be so low in the specimen that using conventional methods a tested portion will not reveal microbial presence. Recently, improvements useful for detecting low concentrations of microorganisms have been disclosed which have greatly improved detection of septicemia in blood before microorganisms have proliferated to such an extent that the patient is in a severe disease state.
Recently developed method and apparatus for concentrating and detecting microorganisms from a sample fluid are disclosed in U.S. Pat. No. 4,131,512 entitled "Method of Detecting Microbial Pathogens Employing a Cushioning Agent" and its division, U.S. Pat. No. 4,212,948 entitled "Apparatus For Detecting Microbia Pathogens Employing A Cushioning Agent". The technique disclosed in the above patents involves (when analyzing a blood sample) pre-lysis of corpuscular compounds followed by centrifugation to concentrate the microorganisms away from the other constituents including antimicrobial factors present in the blood. The concentrated microorganisms are then placed upon a nutrient media such that substances inhibitory to microbial growth present in the sample is diluted a minimum of sixty-fold. It has been previously documented that this technique yielded more positive cultures than the conventional liquid broth culture, the pour plate method, or the filtration method using the solid matrix filter. Gordon Dorn, Geoffrey A. Land, and George E. Wilson, "Improved Blood Culture Technique Based on Centrifugation: Clinical Evaluation," 9 J. Clinical Microbiology 391-396 (1979).
A problem remains in the field of microbial analysis despite the increasing sophistication in techniques for detecting and determining the identity of microorganisms within a specimen because the accuracy of the techniques is limited by the microbial integrity of the sample analyzed. By "microbial integrity" it is meant that a specimen taken at one point in time (t.sub.O) and analyzed at another point in time (t.sub.l) will provide an accurate representation of the microbial population of interest in the patient, food supply or other source from which the specimen was taken, when the specimen is analyzed.
At least three major factors exist which contribute to the lack of microbial integrity of specimens at t.sub.l. The first is that specimens often contain antimicrobial factors which may kill microorganisms of interest before t.sub.l. A second factor is microorganisms of interest may not survive in the specimen until t.sub.l even if no antimicrobial factors are present. Third, certain microorganisms may reproduce much more rapidly in a specimen than, for example, in the patient from whom the specimen was taken. Fast-growing but relatively harmless or irrelevant microorganisms may overwhelm the specimen so that more harmful species of interest are not detected by the analyzing laboratory. Failure to detect the important organism causes misinterpretation of the contamination problem even though the laboratory may correctly identify the organisms that have proliferated. In each case, the sample analyzed at t.sub.l will not give an accurate picture of the microbial problem in the patient or other source. Since drug therapy prescribed by a physician may be dependent on laboratory determinations of type of infecting microorganisms and degree of infection, solving the problem of microbial integrity may be vital to the recovery of the patient. False negatives with respect to food processing equipment or food itself may be detrimental to public health. In addition, misidentification of contamination in the food-related area may prevent discovering the source of contamination or cause the needless disposal of products. Discovering the source is often necessary to prevent future incidents of contamination.
Where antimicrobial factors, such as antibiotic drugs, are present in a specimen several problems arise. For example, a patient given antibiotics by his or her physician may have a level of such drugs in the blood or urine. At t.sub.O, when a urine specimen is taken (for example), the urine may contain living microorganisms and some antibiotic. The antibiotic may continue to work to kill the microorganisms in the specimen so that at t.sub.l, no living microorganisms remain. The laboratory may test the urine specimen and conclude that the patient no longer has a microbial problem However, this may be inaccurate. Unlike the specimen, the patient's system may continue to be seeded with microorganisms from the source of infection. While the level of antibiotics in the specimen might be sufficient to kill microorganisms therein, this does not necessarily reflect the status of the infection within the patient. Additionally, living organisms are required for identification and antibiotic susceptibility testing of microorganisms. If the specimen arriving at the laboratory has no living microorganisms, the laboratory cannot usually accurately identify the organisms nor determine antibiotic susceptibility. Drugs which may be more effective in eliminating particular organisms may not be prescribed if a less effective drug is taken by a patient and is effective enough to destroy the microbial integrity of the specimen taken from that patient, even though it is not effective enough in the patient's system to destroy the infecting microorganisms Natural bacteriocidal substances found in some specimens, such as blood, may also change the microbial integrity of the specimen before it is analyzed causing inaccurate results.
Even if no antimicrobial factors are present in a specimen, a microbial integrity problem remains If living microorganisms are contained in a specimen at t.sub.O, but fail to survive to t.sub.l, no microorganisms will likely be detected by the laboratory because detection techniques are chiefly based on microorganism reproduction. Such a situation will lead to false negative reports and potentially harmful consequences if microbial infections or contaminations go untreated.
Organisms may reproduce so well in a specimen that laboratory analysis will falsely indicate that the patient, foodstuff, or food processing equipment is highly contaminated. Incorrect drug therapy may be administered that is both unnecessary and potentially harmful by itself to some patients. Also, the rapidly-reproducing organism may cause other more harmful microorganisms in the specimen to die in the specimen, although they may be reproducing rapidly in the patient. Since appropriate drug therapy may differ depending on the identity of the problem organism, the patient may not be treated properly for eliminating the more virulent, undetected microorganism and will thus be harmed. In the case of food analysis, misidentification of the source of contamination may result and thus the source which introduced the virulent microorganism may not be discovered.
The problem of lack of microbia integrity in specimens may be increased because of hospital inefficiency in transporting the specimen to the laboratory and backlogs occurring in the laboratory of samples to be analyzed Although most textbooks and handbooks of microbiological technique mandate a specimen hold time of less than two hours, it is often impractical to comply with this standard of efficiency. The problem may be even worse when the specimen must be transported from a remote site such as a doctor's office, a food processing plant, or a sewage-treatment plant to a central laboratory. The accuracy of analysis decreases the longer it takes to transport the specimen to the laboratory because of the deterioration of microbial integrity of the specimen.
Thus, ideally, when testing fluids for differing levels of organisms, the level of organisms present in the sample should not change between the time a sample is taken, and the time the sample is tested. However, the environment of a test sample is usually different from the environment of the tested fluid due to varying temperature, light, availability of nutrients, etc. This can cause large differences in the number of organisms present at the time of testing. For example, the number of live bacteria in a urine sample which has been out of the donor's body for a few hours can increase due to reproduction, or they can decrease due to the action of antibiotics which the donor may be taking. Likewise, organisms present in water can quickly consume all available nutrients in a sample container and die before a test can be conducted. In any case, the longer the time between taking the sample and testing the sample, the higher probability of an incorrect test result there will be.
While the specimen quality problem has been addressed by the art, no known approach has been entirely effective and some have introduced further problems.
The simplest approach disclosed by the prior art is rapid transfer from the point of specimen collection to the point of analysis. For organisms particularly sensitive to transport, immediate streaking on nutrient plates has been suggested literally at the bedside of the patient. As pointed out, it is often difficult to make sure that a specimen has been transported within a recommended time frame. Even if it has, if the specimen contains antibiotics, up to 50% of the microorganisms of interest may be killed within 15-20 minutes. Thus, it can be seen that transport to a lab in two hours or less may be insufficient. Immediate streaking at bedside may cause loss of aseptic technique and the remaining problem of transport of the plate to the laboratory. Antibiotic presence may still present a problem.
The transport of specimens in the past has often been undertaken in initially sterile containers in an attempt to improve specimen quality. Even if a specimen is collected in a sterile container, however, the microbial integrity of the specimen may deteriorate during transport because initial container sterility neither prevents death nor overgrowth of microbes in the specimen. Additionally, sterility of containers could be lost where such specimens as urine, for example, are collected as soon as the closure means is removed for micturition.
In U.S. Pat. No. 4,145,304 ('304) and U.S. Pat. No. 4,174,277 ('277), a method and structures for the removal of antimicrobial factors were disclosed. A mixed resin bed adsorbs the antibiotics to prevent cidal effects on the microorganisms of interest. Multiple physical entries into the specimen are required in the resin bed system in that the specimen must be collected from the patient, transferred to the resin bed for adsorption of antibiotics, and removed from the resin bed. The more physical entries a specimen is subjected to, the higher the risk of microbial contamination from the skin of the operator or the environment. The resin bed is insoluble and therefore requires physical manipulations before the specimen may be analyzed. Loss of microorganisms may result from some non-selective adsorption. Additionally, the mixed resin system fails to address the maintenance of microbial cells in a viable condition without replication.
Certain systems are taught for use in urine specimens which address the problem of uncontrolled growth of particular species of interest which could skew analysis. However, most of these systems focus on killing bacteria that may be present since the specimen will be assayed for general chemical levels, such as glucose, bilirubin etc. In systems taught for preserving microbial integrity, antibiotic blockage is generally not addressed. Thus, no means of preserving the actual count of microorganisms in the presence or absence of bactericidal agents is addressed by known urine specimen-treating agents.
Maintaining a specimen at about 4.degree. C. from the time of collection to the time of analysis is another known approach to attempting to maintain specimen quality Since low temperature may slow microbial growth, antibiotics which act on only replicating organisms may lose effectiveness. However, this approach is impractical in the field, and the low temperature may detrimentally affect the viability of certain microorganisms while being an ineffective control on the growth of others. Additionally, the action of antibiotics is not necessarily controlled by the low-temperature approach. An example of a microorganism which may be killed by the cold is Streptococcus pneumoniae, one that a physician would be interested in detecting as it is an etiological agent of lobar pneumonia disease. Thus, it is preferable to maintain the sample at room temperature of about 21-25.degree. C.
Other methods for improving specimen quality include Amies (C. Amies and F. Path, 58 Canadian J. Public Health 296 (1967)) and Stuarts (R. Stuart et. al., "The Problem of Transport of Specimens For Culture of Gonococci," 45 Canadian J. Public Health 73 (1954)). These methods may provide some improvement of specimen quality for some microorganisms of interest, however these systems fail to address the possible presence of antibiotics in a specimen, the differing nutritional needs of different microorganisms, and the effect of specimen hold time on accurate microorganism quantitation.
Another problem left unaddressed by previous approaches to microbial detection is the possibility that additional microorganisms will be introduced to a specimen from an external source. This "contamination" of the specimen will cause inaccurate results since, for example, a patient may be deemed to have a microorganism in the blood that in fact is not present. Contamination of specimens becomes more likely the more the specimen is transferred from container to container and the more it undergoes physical manipulations. For example, a commercially available system for urine specimen transport (Becton-Dickenson) requires manipulation from the urine collection vessel to the container with the preservative therein. It is therefore desirable to provide collection vessels which reduce the manipulations required, provide a means to instantly preserve the microbial integrity of a sample, and in a most preferred embodiment can be utilized for other processing steps in the analysis of microorganisms of interest.
Therefore, a method and means is needed for receiving a fluid sample suspected of containing microbial pathogens and antimicrobial factors which will minimize the risk of contamination, reduce or eliminate the requirement of sterility of the collection vessel for some specimens, provide for deactivation of antimicrobial factors during the time that the sample is transported so that once the sample is removed from the collection and/or processing vessel and placed on growth media, the microorganisms of interest present in the sample including the fastidious microorganisms of interest will proliferate and become identifiable, and which will maintain the viability of at least some of the microorganisms of interest, preferably so that the microbial integrity of the sample is maintained from time of specimen collection (t.sub.O) to the time of specimen analysis (t.sub.l).
It has now been found that microbial integrity of patient specimens and other specimens may be preserved so that analysis at a t.sub.l up to about 72 hours after t.sub.O will result in a much more accurate representation of the microbial population in that sample than has previously been possible. This has been done by providing an admixture of individual chemicals which solubilize in an aqueous specimen to form a unique mixture which acts synergistically as a preservative of microbial integrity of the specimen. By "preservative" it is meant that the unique mixture prevents replication of microorganisms of interest, allows improved survival of said microorganisms until the inception of laboratory analysis, and blocks the action of antimicrobial factors that may be present in the specimen. By "microorganisms of interest" it is meant the microorganisms to be tested for in the laboratory protocol. It may not be necessary or desirable to preserve the viability, for example, of every possible microorganism that may be present in a given specimen. In the food industry, for example, non-harmful or even beneficial microorganisms may be present in food which a laboratory would not be interested in identifying. However, the laboratory would be interested in testing for microorganisms potentially harmful to human health. Therefore, preservation of the latter "microorganisms of interest" would be addressed by the present invention. In addition, the growth of the microorganisms which are not of interest must be kept in check to prevent masking of the harmful microorganism in the analysis procedure, and to prevent the rapidly producing non-harmful organisms from depleting the nutrients and causing death of other microbes. The present invention is effective in inhibiting replication of such potentially interfering organisms. The present invention thus allows a longer time to elapse between specimen collection and specimen analysis than has previously been possible without sacrificing accuracy. It also allows for more accurate analysis even if a sample is analyzed within a short time period because it blocks the action of antimicrobial factors which may destroy microorganisms of interest even within the two hour processing time period recommended in the prior art.
In addition, no reason is known why the disclosed specimen transport system would not be advantageous for improving the accuracy of analysis of specimens for periods exceeding 72 hours. If the viability of even a few microorganisms of interest is maintained, the microbial integrity of specimen analyzed will be improved over that possible according to the prior art, resulting in improved laboratory analysis.
Disclosed is a novel method, article and compositions for detecting microbial pathogens. In another aspect, this invention relates to a novel technique and means for selectively separating microorganisms from a sample fluid which contain antimicrobial factors. In still another aspect, this invention relates to a method and means for use in the detection of microbial pathogens which provides improved recovery of microorganisms. In yet another aspect, this invention relates to a method and means for accurately quantitating the number of microorganisms present in a sample fluid at a given time when quantitated at a later time.
An article for receiving specimens is disclosed which includes a means for preserving the microbial integrity of the specimen.